Streptococcus pneumoniae is an important cause of otitis media, meningitis, bacterernia and pneumonia. Despite the use of antibiotics and vaccines, the prevalence of pneumococcal infections has declined little over the last twenty-five years.
It is generally accepted that immunity to Streptococcus pneumoniae can be mediated by specific antibodies against the polysaccharide capsule of the pneumococcus. However, neonates and young children fail to make an immune response against polysaccharide antigens and can have repeated infections involving the same capsular serotype.
One approach to immunizing infants against a number of encapsulated bacteria is to conjugate the capsular polysaccharide antigens to proteins to make them immunogenic, This approach has been successful, for example, with Haemophilus influenzae b (see U.S. Pat. No. 4,496,538 to Gordon and U.S. Pat. No. 4,673,574 to Anderson). However, there are over eighty known capsular serotypes of S. pneumoniae of which twenty-three account for most of the disease. For a pneumococcal polysaccharide-protein conjugate to be successful, the capsular types responsible for most pneumococcal infections would have to be made adequately immunogenic. This approach may be difficult, because the twenty-three polyesaccharides included in the presently-available vaccine are not all adequately immunogenic, even in adults, Furthermore, such a vacine would probably be much more expensive to produce than any of the other childhood vaccines in routine use.
An alternative approach for protecting children, and also the elderly, from pneumococcal infection would be to identify protein antigens that could elicit protective immune responses. Such proteins may serve as a vaccine by themselves, may be used in conjunction with successful polysaccharide-protein conjugates, or as carriers for polysaccharides.
In McDaniel et al (I), J.Exp.Med. 160:386-397, 1984, there is described the production of hybridoma antibodies that recognize cell surface proteins on S. pneumoniae and protection of mice from infection with certain strains of encapsulated pneumococci by such antibodies. This surface protein antigen has been termed "pneumococcal surface protein A" or PspA for short.
In McDaniel et al (II), Microbial Pathogenesis 1:519-531, 1986, there are described studies on the characterization of the PspA. From the results of McDaniel (II), McDaniel (III), J.Exp.Med. 165:381-394, 1987, Waltman et al., Microb. Pathog. 8:61-69, 1990 and Crain et al., Infect. Immun. 58:3293-3299, 1990, it was also apparent that the PspAs of different strains frequently exhibit considerable diversity in terms of their epitopes, and apparent molecular weight.
In McDaniel et al (III), there is disclosed that immunization of X-linked immunodeficient (XID) mice with non-encapsulated pneumococci expressing PspA, but not isogenic pneumococci lacking PspA, protects mice from subsequent fatal infection with pneumococci.
In McDaniel et al (IV), infect. Immun., 59:222-228, 1991, there is described immunization of mice with a recombinant full length fragment of PspA that is able to elicit protection against pneumococcal strains of capsular types 6A and 3.
In Crain et al, (supra) there is described a rabbit antiserum that detects PspA in 100% (n=95) of clinical and laboratory isolates of strains of S. pneumoniae. When reacted with seven monoclonal antibodies to PspA, fifty-Seven S. pneumoniae isolates exhibited thirty-one different patterns of reactivity. Accordingly, although a large number of serologically-different PspAs exist, there are extensive cross-reactions between PspAs.
The PspA protein type in independent of capsular type. It would seem that genetic mutation or exchange in the environment has allowed for the development of a large pool of strains which are highly diverse with respect to capsule, PspA, and possibly other molecules with variable structures. Variability of PspA's from different strains also is evident in their molecular weights, which range from 67 to 99 kD. The observed differences are stably inherited and are not the result of protein degradation.
Immunization with an induced isolate of E. coli infected with a recombinant .lambda. gt11 clone producing PspA, elicited protection against challenge with several S. pneumoniae strains representing different capsular and PspA types, as described in. The protection was assumed to be the result of immunization with the recombinant PspA, since a .lambda. gt II phage lacking the pspA gene failed to elicit protection. McDaniel et al (IV), Infect. Immun. 59:222-228, 1991. Although clones expressing PspA were constructed according to that paper, the product was in low yield and unstable, and isolation or purification from cell wall, cytoplasmic and medium components following lysis was not effected.
While the protein is variable in structure between different pneumococcal strains, numerous cross-reactions exist between all PsPA's, suggesting that enough common epitopes may be present to allow a single PspA or at most a small number of PspA's to elicit protection against a large number of S. pneumoniae strains.
In addition to the published literature specifically referred to above, the inventors, in conjunction with co-workers, have published further details concerning PspA's as follows:
1. Abstracts of 89th Annual Meeting of the American Society for Microbiology, p. 125, item D-257, May 1989; PA1 2. Abstracts of 90th Annual Meeting of the American Society for Microbiology, p. 98, item D-106, May 1990; PA1 3. Abstracts of 3rd International ASM Conference on Streptococcal Genetics, p. 11, item 12, June 1990; PA1 4. Talkington et al, Infect. Immun. 59:1285-1289, 1991; PA1 5. Yother et al (I), J. Bacteriol. 174:601-609, 1992; PA1 6. Yother et al (II), J. Bacteriol. 174:610-618, 1992, and PA1 7, McDaniel et al (V), Microbiol Pathogenesis, 13:261-268, 1992.
In the aforementioned copending United States patent applications Ser. No. 656,773 and 835,698 (corresponding to published International patent application, WO 92/1448), as well as in Yother et al (I) and (II), the disclosures of which are incorporated herein by reference, there are described the preparation of mutants of S. pneumomiae that secrete an immunogenic truncated form of the PspA protein, and the isolation and purification of the secreted protein. The truncated form of PspA was found to be immunoprotective and to contain the protective apitopes of PspA. The PspA protein described therein is soluble in physiologic solution and lacks at least the functional cell membrane anchor region.
The aforementioned USSNs' also describe characteristics of the mature Rx1 PspA protein. The mature protein in composed of 588 amino acids and has a molecular weight of 65 kD. The N-terminal 288 amino acids are highly charged and predict an a-helical coiled-coil protein structure. The C-terminal 217 amino acids contain the surface anchor of PspA and does not appear to be .alpha.-helical. In the middle of the molecule is a proline rich region that in thought to traverse the cell wall.
In the specification which follows and the drawings accompanying the same, there are utilized certain accepted abbreviations with respect to the amino acids represented thereby. The following Table I identifies those abbreviations:
TABLE I ______________________________________ AMINO ACID ABBREVIATIONS ______________________________________ A = Ala = Alanine M = Met = Methionine C = Cys = Cysteine N = Asn = Asparagine D = Asp = Aspartic Acid P = Pro = Proline E = Glu = Glutamic Acid Q = Gln = Glutamine F = Phe = Phenylalanine R = Arg = Arginine G = Gly = Glycine S = Ser = Serine H = His = Histidine T = Thr = Threonine I = Ile = Isoleucine V = Val = Valine K = Lys = Lysine W = Try = Tryptophan L = Leu = Leucine Y = Tyr = Tyrosine ______________________________________